GC–MS Characterization and Biological Evaluation of Costus speciosus Rhizomes: Antibacterial Activity and Mechanism of Action against MDR and XDR Bacteria with Anticancer Potential

The rapid emergence of multidrug-resistant (MDR) and extensively drug-resistant (XRD) bacteria represents a major global health challenge, necessitating the exploration of novel antimicrobial agents from natural sources. This study investigated the antibacterial potential Costus speciosus rhizome extracts and evaluated the cytotoxic activity of the most active fraction. Rhizomes were extracted using ethanol, methanol, ethyl acetate, and cold water. Antimicrobial susceptibility testing was performed on 29 bacterial isolates from clinical specimens and meat products to determine resistance profiles. Based on resistance patterns, 11 representative MDR/XDR isolates, including Staphylococcus aureus , Escherichia coli , Klebsiella pneumoniae , Serratia rubbidea , and Hafnia alvei , were selected for further evaluation using agar well diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays. The ethyl acetate extract showed the strongest antibacterial activity and was subsequently fractionated using column chromatography, yielding 43 fractions. Fractions were screened for antibacterial activity, and the most active fraction was further analyzed by gas chromatography–mass spectrometry (GC–MS). Transmission electron microscopy (TEM) was used to investigate the antibacterial mode of action, while cytotoxicity was assessed against HepG2, A-549, and WI-38 cell lines. The ethyl acetate extract showed the highest antibacterial activity, whereas the cold-water extract exhibited weak activity. Among the 43 fractions, nine displayed antibacterial activity, with fraction 4 exhibiting the strongest effect. GC–MS analysis of fraction 4 identified major compounds, including azuleno[4,5-b]furan-2(3H)-one (65.87%), dihydrodehydrocostus lactone (10.54%), and (Z,Z,Z)-1,8,11,14-heptadecatetraene, (9.91%). The MIC and MBC values ranged from 0.062 − 1.5 mg/mL and 0.25 − 1.5 mg/mL, respectively. TEM revealed severe cellular damage, including cell wall disruption and cytoplasmic leakage. The active fraction demonstrated potent cytotoxic activity against HepG2 and A-549 cell lines, with IC ₅₀ values of 5.17 ± 0.49 and 8.27 ± 0.61 µg/mL, respectively, and a CC ₅₀ value of 23.92 ± 0.86 µg/ml against WI-38 cells, yielding selectivity indices (SI) of 4.63 and 2.89, respectively. In conclusion, the ethyl acetate fraction of Costus speciosus represents a promising source of antibacterial and anticancer agents against drug-resistant pathogens.